Alkylated derivatives of antibiotic BM123γ

ABSTRACT

This disclosure describes a novel series of potent antibacterial agents derived by reductive alkylation of antibiotic BM123γ with certain classes of aldehydes and ketones.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of our copending applicationSer. No. 612,975, filed Sept. 12, 1975, which in turn is acontinuation-in-part of application Ser. No. 529,862, filed Dec. 5,1974, both now abandoned.

BRIEF SUMMARY OF THE INVENTION

This invention relates to a novel group of antibiotics and, moreparticularly, is concerned with a novel series of potent antibacterialagents derived by reductive alkylation of antiobiotic BM123γ with analdehyde or ketone of the following general formulae: ##STR1## whereinR₁ is hydrogen, lower alkyl, halo substituted lower alkyl, loweralkenyl, phenyl, monosubstituted phenyl, phenyl lower alkyl, 2-furyl,methyl substituted 2-furyl, 2-thienyl, methyl substituted 2-thienyl,2-pyrryl, methyl substituted 2-pyrryl, 2-pyridyl or 2-quinolyl; R₂ islower alkyl, halo substituted lower alkyl or phenyl lower alkyl; R₃ islower alkyl, halo substituted lower alkyl, lower alkenyl, lowercycloalkyl, phenyl, monosubstituted phenyl, phenyl lower alkyl ormonosubstituted phenyl lower alkyl; and R₂ and R₃ taken together withthe associated carbonyl group is cyclopentanone, mono-lower alkylsubstituted cyclopentanone, di-lower alkyl substituted cyclopentanone,tri-lower alkyl substituted cyclopentanone, cyclohexanone, mono-loweralkyl substituted cyclohexanone, di-lower alkyl substitutedcyclohexanone or tri-lower alkyl substituted cyclohexanone. Suitablelower alkyl and halo substituted lower alkyl groups contemplated by thepresent invention are those having up to six carbon atoms wherein halois exemplified by chloro, bromo, and iodo such as methyl, ethyl,isopropyl, sec-butyl, n-amyl, dichloromethyl, 2-bromoethyl,2,3-diiodopropyl, γ-chloropropyl, etc. Suitable lower alkenyl groups arethose having up to four carbon atoms such as vinyl, allyl, propenyl,isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, isobutenyl, etc. Suitablelower cycloalkyl groups are cyclopenyl, cyclohexyl, and cycloheptyl.Suitable monosubstituted phenyl groups contemplated by the presentinvention are, for example, p-acetamidophenyl, m-nitrophenyl,m-mercaptophenyl, o-anisyl, p-anisyl, o-tolyl, p-tolyl, and the likewhereas phenyl lower alkyl is exemplified by benzyl, α-phenylethyl, andβ-phenylethyl. Suitable monosubstituted phenyl lower alkyl groups may beo, m, or p-chlorobenzyl, α-(p-aminophenyl)ethyl, β-(m-nitrophenyl)ethyl,etc. Suitable methyl substituted 2-furyl, 2-thienyl, and 2-pyrryl groupswhich may be employed are, for example, 5-methyl-2-furyl,3,4-dimethyl-2-furyl, 4-methyl- 2-thienyl, 3,5-dimethyl-2-thienyl,5-methyl-2-pyrryl, 1,3,4-trimethyl-2-pyrryl, and the like.

The reductive alkylation process whereby the novel antibacterial agentsof the present invention may be prepared is carried out as follows.Antibiotic BM123γ, BM123γ₁, or BM123γ₂ is dissolved in a suitablesolvent such as water, methanol, methyl cellosolve, or mixtures thereof,an amount in excess of an equimolar amount of the desired aldehyde orketone is then added followed by the addition of a reductive sufficiencyof sodium cyanobrorohydride. The pH of the reaction mixture ismaintained at 6.0-8.0 with dilute mineral acid during the course of thereaction. After one to 24 hours at ambient temperature (10°-35° C.), thereaction mixture is evaporated to dryness in vacuo and the residue istriturated with methanol and filtered. The filtrate is diluted withacetone and the solid product that precipitates is removed by filtrationand dried in vacuo.

Aldehydes which may be so employed in the above process are, forexample, acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde,crotonaldehyde, valeraldehyde, benzaldehyde, p-cyanobenzaldehyde,salicylaldehyde, cinnamaldehyde, trichloroacetaldehyde, etc. Ketoneswhich may be so employed in the above process are, for example, acetone,2-butanone, 1,3-dibromoacetone, chloroacetone, acetophenone,m-chloroacetophenone, p-bromoacetophenone,p-trifluoromethylacetophenone, m-nitroacetophenone,p-dimethylaminoacetophenone, etc.

The products are obtained from the reductive alkylation reactionmixtures by standard procedures such as precipitation, concentration,solvent extraction or combinations of these procedures. After isolation,the products may be purified by any of the generally known methods forpurification. These include recrystallization from various solvents andmixed solvent systems, chromatographic techniques, and counter currentdistribution, all of which are usually employed for this purpose.

The novel antibacterial agents of the present invention are organicbases and thus are capable of forming acid-addition salts with a varietyof organic and inorganic salt-forming reagents. Thus, acid-additionsalts, formed by admixture of the antibacterial free base with up tothree equivalents of an acid, suitably in a neutral solvent, are formedwith such acids as sulfuric, phosphoric, hydrochloric, hydrobromic,sulfamic, citric, maleic, fumaric, tartaric, acetic, benzoic, gluconic,ascorbic, and related acids. The acid-addition salts of theantibacterial agents of the present invention are, in general,crystalline solids relatively soluble in water, methanol and ethanol butare relatively insoluble in non-polar organic solvents such as diethylether, benzene, toluene, and the like. For purposes of this invention,the antibacterial free bases are equivalent to their non-toxic acidaddition salts.

DETAILED DESCRIPTION OF THE INVENTION

The antibiotics designated BM123β₁, BM123β₂, BM123γ₁ and BM123γ₂ areformed during the cultivation under controlled conditions of a newstrain of an undetermined species of Nocardia. This new antibioticproducing strain was isolated from a garden soil sample collected atOceola, Iowa, and is maintained in the culture collection of the LederleLaboratories Division, American Cyanamid Company, Pearl River, N.Y. asCulture No. BM123. A viable culture of the new microorganism has beendeposited with the Culture Collection Laboratory, Northern UtilizationResearch and Development Division, United States Department ofAgriculture, Peoria, Illinois, and has been added to its permanentcollection. It is freely available to the public in this depositoryunder its accession No. NRRL 5646. Herein BM123β refers to a mixture inany proportion of BM123β₁ and BM123β₂ , and BM123γ refers to a mixturein any proportion of BM123γ₁ and BM123γ₂.

The following is a general description of the microorganism Nocardiasp., NRRL 5646, based on diagnostic characteristics observed.Observations were made of the cultural, physiological, and morphologicalfeatures of the organism in accordance with the methods detailed byShirling and Gottlieb, Internat. Journ, of Syst, Bacteriol. 16:213-240(1966). The chemical composition of the culture was determined by theprocedures given by Lechevalier et al., Advan. Appl. Microbiol. 14:47-72(1971). The underscored descriptive colors and color chip designationsare taken from Jacobson et al., Color Harmony Manual, 3rd ed. (1948),Container Corp. of America, Chicago, Illinois. Descriptive details arerecorded in Tables I through V below.

Amount of Growth

Moderate on yeast extract, asparagine dextrose, Benedict's, Bennett'spotato dextrose and Weinstein's agars; light on Hickey and Tresner's,tomato paste, oatmeal, and pablum agars and a trace of growth oninorganic salts-starch, Kuster's oatflake, Czapek's solution, and riceagars.

Aerial Mycelium

Aerial mycelium whitish when present; produced only on yeast extract,asparagine dextrose, Benedict's, Bennett's and potato dextrose agars.

Soluble Pigments

No soluble pigments produced.

Reverse Color

Colorless to yellowish shades.

Miscellaneous Physiological Reactions

No liquefaction of gelatin; nitrates reduced to nitrites in 7 days;melanoid pigments not formed on peptone-iron agar; no peptonization orcurd formation in purple milk; NaCl tolerance in yeast extract agar ≦ 4%but ≦ 7%; optimal growth temperature 32° C. Carbon source utilization,according to the Pyridham and Gottlieb method [J. Bacteriol. 56:107-114(1948)] as follows: Good utilization of glycerol, salicin, d-trehaloseand dextrose; fair utilization of i-inositol; and poor tonon-utilization of d-fructose, maltose, adonitol, 1-arabinose, lactose,d-mannitol, d-melibiose, d-raffinose, 1-rhamnose, sucrose and d-xylose.

Chemical Composition

The organism belongs to cell wall type IV, i.e., containsmeso-2,6-diaminopimelic acid and has a type A whole-cell sugar pattern,i.e., contains arabinose and galactose. methylated whose cell extracts,when subjected to gas chromatography, showed fatty acid patterns similarto those produced by Nocardia asteroides ATCC 3308.

Micromorphology

Aerial mycelium arises from substrate mycelium as sparingly branchedmoderately long flexuous elements that commonly terminate in elongatedprimitive spirals. The flexuous elements are irregularly segmented intoshort elliptical to cyclindrical sections (spores) which disarticulatereadily. The spiral terminal portions are less conspicuously segmented.Segments generally range 0.8-1.7 μm × 0.3-0.5 μm, averaging 0.4 μm × 1.2μm.

Diagnosis

The morphological characteristics of Culture No. BM123 are difficult toobserve and interpret because of the poor development of aerial myceliumon most media. Hence, considerable importance is attached, out ofnecessity, to the chemical analysis in determining the genericrelationship of the organism. On the basis of the system proposed byLechevalier et al., Culture No. BM123 contains meso-2,6-diaminopimelicacid in its whole cells and sugar analysis shows arabinose and galactoseto be present. Therefore, the culture belongs to cell wall type IV. Acomparison of the gas chromatography pattern of Culture No. BM123 withthat of Nocardia asteroides ATCC 3308 showed the two to be remarkablysimilar. Other characteristics of Culture No. BM123 that are in keepingwith the Nocardia concept, are its fragmenting aerial growth on somemedia and the total absence of aerial growth on most media. In view ofthe lack of adequate criteria for the characterization of Nocardia tothe species level, no attempt has been made to make this determination.Therefore, Culture No. BM123 will be considered an undetermined speciesof Nocardia until such a diagnosis is feasible.

                                      TABLE I                                     __________________________________________________________________________    Cultural Characteristics of Nocardia sp. NRRL 5646                            __________________________________________________________________________    Incubation: 14 days             Temperature: 32° C.                    __________________________________________________________________________                Amount of                                                                            Aerial Mycelium                                                                            Soluble                                                                            Reverse                                  Medium      Growth And/Or Spores                                                                              Pigment                                                                            Color   Remarks                          __________________________________________________________________________    Yeast Extract Agar                                                                        Moderate                                                                            Aerial mycelium whitish,                                                                    None  Mustard                                                                            Darkened areas in sub-                               light                    strate mycelium.                                                        (3 ie)                                                                              Coremia formed on sur-                                                        face mycelium                      Hickey and Tresner's                                                                      Light No aerial mycelium                                                                          None Colorless                                                                           Peripheral areas of                Agar                                 to    colonies becoming                                                       Yellowish-                                                                          olive-green                                                             green                                    Asparagine dextrose                                                                       Moderate                                                                            Trace of whitish aerial                                                                     None Amber Surface lightly                    Agar              mycelium           (3 lc)                                                                              wrinkled                           Benedict's Agar                                                                           Moderate                                                                            Aerial mycelium whitish                                                                     None Nude Tan                                                                            Coremia abundantly                                   light              (4 gc)                                                                              formed on surface                                                             mycelium                           Bennett's Agar                                                                            Moderate                                                                            Trace of whitish aerial                                                                     None Camel Surface lightly                                      mycelium           (3 ie)                                                                              wrinkled                           Inorganic Salts-                                                                          Trace No aerial mycelium                                                                          None Colorless                                starch Agar                                                                   Kuster's Oatflake                                                                         Trace No aerial mycelium                                                                          None Colorless                                Agar                                                                          Czapek's Solution                                                                         Trace No aerial mycelium                                                                          None Colorless                                Agar                                                                          Potato dextrose                                                                           Moderate                                                                            Aerial mycelium whitish,                                                                    None Camel                                    Agar              light              (3 ie)                                   Tomato Paste                                                                              Light No aerial mycelium                                                                          None Colorless                                Oatmeal Agar                                                                  Pablum Agar Light No aerial mycelium                                                                          None Colorless                                Rice Agar   Trace No aerial mycelium                                                                          None Colorless                                Weinstein's Agar                                                                          Moderate                                                                            No aerial mycelium                                                                          None Colorless                                                                     to                                                                            yellowish                                __________________________________________________________________________

                  TABLE II                                                        ______________________________________                                        Micromorphology of Nocardia sp. NRRL 5646                                     ______________________________________                                                     Aerial Mycelium and/or Sporiferous                               Medium          Structures                                                    ______________________________________                                        Yeast Extract                                                                              Aerial mycelium arises from sub-                                 Agar         strate mycelium as sparingly                                                  branced, flexous elements that                                                commonly terminate in elongated                                               primitive spirals. The flexuous                                               elements are irregularly segmented                                            into short sections (spores?)                                                 which disarticulate readily. The                                              spiral terminal portions are less                                             conspicuously segmented. Segments                                             generally range 0.8-1.7 μm ×                                         0.3-0.5 μm, averaging 0.4 μm ×                                    1.2 μm                                                        ______________________________________                                    

                                      TABLE III                                   __________________________________________________________________________    Miscellaneous Physiological Reaction of Nocardia sp. NRRL                     __________________________________________________________________________    5646                                                                          Medium     Incubation Period                                                                        Amount of Growth                                                                        Physiological Reaction                        __________________________________________________________________________    Gelatin     7 days    Light     No liquefaction                               Gelatin    14 days    Good      No liquefaction                               Organic Nitrate                                                                           7 days    Good      Nitrates reduced to nitrites                  Broth                                                                         Organic Nitrate                                                                          14 days    Good      Nitrates reduced to nitriles                  Broth                                                                         Peptone-iron                                                                             24-48 hours                                                                              Good      No melanin pigments reduced                   Agar                                                                          Purple Milk                                                                               7 days    Good      No peptonization or curd formation            Yeast extract                                                                             7 days    Moderate  NaCl tolerance ≧  4 % but ≦                                      7 %                                          Agar plus (4, 7,                                                              10 and 13%) NaCl                                                              __________________________________________________________________________

                  TABLE IV                                                        ______________________________________                                        Carbon Source Utilization Pattern of Nocardia sp. NRRL 5646                   ______________________________________                                        Incubation: 10 days                                                                              Temperature: 32° C.                                 ______________________________________                                        Carbon Source      Utilization*                                               ______________________________________                                        Adonitol           0                                                          1-Arabinose        0                                                          Glycerol           3                                                          d-Fructose         1                                                          i-Inositol         2                                                          Lactose            0                                                          d-Mannitol         0                                                          Salicin            2                                                          d-Melibiose        0                                                          d-Raffinose        0                                                          Rhamnose           0                                                          Maltose            1                                                          Sucrose            0                                                          d-Trehalose        3                                                          d-Xylose           0                                                          Dextrose           3                                                          Negative Control   0                                                          ______________________________________                                         *3-Good Utilization                                                           2-Fair Utilization                                                            1-Poor Utilization                                                            0-No Utilization                                                         

                  TABLE V                                                         ______________________________________                                        Chemical Composition of Nocardia sp. NRRL 5646                                ______________________________________                                        Cell Wall Type   Major Constituents                                           ______________________________________                                        Type IV          meso-DAP, arabinose,                                                          galactose                                                    ______________________________________                                    

The production of BM123β and BM123γ is not limited to this particularorganism or to organisms fully answering the above growth andmicroscopic characteristics which are given for illustrative purposesonly. In fact, mutants produced from this organism by various means suchas exposure to X-radiation, ultra-violet radiation, nitrogen mustard,actinophages, and the like, may also be used. A viable culture of atypical such mutant strain has been deposited with the CultureCollection Laboratory, Northern Utilization Research and DevelopmentDivision, United States Department of Agriculture, Peoria, Illinois, andhas been added to its permanent collection under its accession numberNRRL 8050. Although the cultural, physiological, and morphologicalfeatures of NRRL 8050 are substantially the same as those of NRRL 5646,it produces enhanced amounts of BM123γ during aerobic fermentation.Also, NRRL 8050 varies from the parent NRRL 5646 as follows:

a. slower reduction of nitrates to nitrites; and

b. production of a rosewood tan mycelial pigment on Bennett's and yeastextract agars.

The novel antibacterial agents of the present invention are, in general,crystalline solids of relatively limited solubility in non-polarsolvents such as diethyl ether and hexane, but considerably more solublein solvents such as water and lower alkanols. Antibiotics BM123γ₁ andBM123γ₂ are structural isomers and may be represented by the followingstructural formulae: ##STR2##

The reductive alkylation of BM123γ, BM123γ₁ or BM123γ₂ with ketonestakes place on the spermadine side-chain to form derivatives of theformula: ##STR3## wherein R is a moiety of the formulae: ##STR4## and R₂and R₃ are as hereinabove defined. The reductive alkylation of BM123γ,BM123γ₁ or BM123γ₂ with aldehydes takes place on the spermadineside-chain to form mono-, di-, and tri-substituted derivatives of theformulae: ##STR5## wherein R and R₁ are as hereinabove defined.

The usefulness of the alkylated derivatives of BM123γ is demonstrated bytheir ability to control systemic lethal infections in mice. These newsubstances show high in vivo antibacterial activity in mice againstEscherichia coli US311 when administered by a single subcutaneous doseto groups of Carworth Farms CF-1 mice, weight about 20 gm., infectedintraperitoneally with a lethal dose of this bacteria in a 10⁻ 3trypticase soy broth TSP dilution of a 5 hour TSP blood culture. InTable VI below is set forth the in vivo activity of typical products ofthis invention (prepared from the indicated carbonyl reagents) againstEscherichia coli US311 in mice. The activity is expressed in terms ofthe ED₅₀ or the dose in mg./kg. of body weight required to protect 50%of the mice against E. coli.

                                      TABLE VI                                    __________________________________________________________________________                                                ED.sub.50 in mg./kg.              Carbonyl Reagent Employed                                                                         Derivative Name         of body weight                    __________________________________________________________________________    1-dipropylamino-2-propanone                                                                       1-methyl-2-(N,N-dipropylamino)-                                                                       0.3                               ethyl-BM123γ                                                            1-chloro-3-pentanone                                                                              1-ethyl-3-chloropropyl-BM123γ                                                                   0.12                              cyclooctanone       cyclooctyl-BM123γ 0.18                              4-methyl-2-pentanone                                                                              1,3-dimethylbutyl-BM123γ                                                                        <0.12                             phenylacetone       1-methyl-2-phenylethyl-BM123γ                                                                   0.18                              trans-4-phenyl-3-buten-2-one                                                                      1-methyl-3-phenylpropen(-2-)yl-                                                                       0.25                              BM123γ                                                                  1-cyclohexyl-1-propanone                                                                          1-cyclohexylpropyl-BM123γ                                                                       0.37                              6-methyl-5-hepten-2-one                                                                           1,5-dimethylhexen(-4-)yl-BM123γ                                                                 0.06                              3-methyl-2-pentanone                                                                              1,2-dimethylbutyl-BM123γ                                                                        0.12                              5-methyl-2-hexanone 1,4-dimethylpentyl-BM123γ                                                                       0.12                              3-ethyl-2-pentanone 1-methyl-2-ethylbutyl-BM123γ                                                                    0.18                              3,5-dimethyl-2-octanone                                                                           1,2,4-trimethylheptyl-BM123γ                                                                    0.37                              3-octanone          1-ethylhexyl-BM123γ                                                                             0.18                              3-methyl-2-hexanone 1,2-dimethylpentyl-BM123γ                                                                       0.18                              3-indolylacetone    1-methyl-2-(β-indolyl)ethyl-                                                                     0.12                                                  BM123γ                                              2-pentanone         1-methylbutyl-BM123γ                                                                            <0.12                             2-butanone          1-methylpropyl-BM123γ                                                                           <0.12                             2-cyclopenten(-1-)yl-acetone                                                                      1-methyl-2-cyclopenten(-2-)-                                                  yl-ethyl-BM123γ   <0.12                             acetone             isopropyl-BM123γ  <0.12                             3-decanone          1-ethyloctyl-BM123γ                                                                             0.25                              3-undecanone        1-ethylnonyl-BM123γ                                                                             0.38                              o-acetoacetotoluidide                                                                             1-methyl-2-o-[tolylcarbamoylethyl]-                                                                   0.25                                                  BM123γ                                              mesityl oxide       1,3-dimethylbuten(-2-)yl-BM123γ                                                                 0.3                               methoxyacetone      1-methyl-2-methoxyethyl-BM123γ                                                                  0.3                               cyclohexylacetone   1-methyl-2-cyclohexylethyl-BM123γ                                                               0.18                              4-(p-hydroxyphenyl)-2-butanone                                                                    1-methyl-3-(4-hydroxyphenyl)ethyl-                                            BM123γ            0.3                               dimethyl(2-oxoheptyl)phosphonate                                                                  1-[(dimethoxyphosphinyl)methyl]-                                              hexyl-BM123γ      0.3                               4-methyl-2-hexanone 1,3-dimethylpentyl-BM123γ                                                                       <0.12                             2,2,4,4-tetramethylcyclopentanone                                                                 2,2,4,4-tetramethylcyclopentyl-BM123γ                                                           0.75                              2,4,4-trimethylcyclopentanone                                                                     2,4,4-trimethylcyclopentyl-BM123γ                                                               0.3                               2-cyclopentylcyclopentanone                                                                       2-cyclopentylcyclopentyl-BM123γ                                                                 0.37                              2-(cyclo-1-hexenyl)cyclohexanone                                                                  2-(1-cyclohexen)cyclohexyl-BM123γ                                                               0.19                              3-tert-pentylcyclopentanone                                                                       3-tert-pentylcyclopentyl-BM123γ                                                                 0.5                               2-cyclohexylcyclohexanone                                                                         2-cyclohexylcyclohexyl-BM123γ                                                                   0.75                              2-ethylcyclohexanone                                                                              2-ethylcyclohexyl-BM123γ                                                                        0.19                              3,3-dimethyl-2-butanone                                                                           1,2,2-trimethylpropyl-BM123γ                                                                    <0.12                             2-undecanone        1-methyldecyl-BM123γ                                                                            >2                                tetrahydrothiopyran-4-one                                                                         4-tetrahydrothiopyranyl-BM123γ                                                                  0.38                              3,5-dimethylcyclohexanone                                                                         3,5-dimethylcyclohexyl-BM123γ                                                                   <0.12                             2-tetradecanone     1-methyltridecyl-BM123γ                                                                         2.0                               1-methoxy-1-buten-3-one                                                                           1-methyl-3-methoxypropen(-2-)yl-                                              BM123γ            >2.0                              4-hydroxy-3-methyl-2-butanone                                                                     1,2-dimethyl-3-hydroxypropyl-BM123γ                                                             0.12                              menthone            3-methyl-6-isopropylcyclohexyl-BM123γ                                                           0.38                              cyclononanone       cyclononyl-BM123γ 0.18                              1-methyl-2-decalone decahydro-1-methyl-2-naphthyl-BM123γ                                                            0.25                              isophorone          3,3-dimethylcyclohexen(-4-)yl-BM123γ                                                            0.37                              3-methyl-2-decalone decahydro-3-methyl-2-naphthyl-BM123γ                                                            0.37                              1-(3,4-dimethoxyphenyl)-2-butanone                                                                2-ethyl-2-(3,4-dimethoxyphenyl)ethyl-                                                                 1.0                                                   BM123γ                                              1-diethylamino-3-butanone                                                                         1-methyl-3-(N,N-diethylamino)propyl-                                          BM123γ            0.18                              ethyl 2-chloroacetoacetate                                                                        1-methyl-2-chloro-2-carbethoxyethyl-                                                                  1.5                               BM123γ                                                                  3-hydroxy-3-methyl-2-butanone                                                                     1,2,2-dimethylhydroxypropyl-BM123γ                                                              0.18                              3-pentanone         1-ethylpropyl-BM123γ                                                                            0.12                              3-methyl-2-butanone 1,2-dimethylpropyl-BM123γ                                                                       0.19                              p-chlorophenylacetone                                                                             1-methyl-2-(4-chlorophenyl)ethyl-                                                                     0.25                                                  BM123γ                                              N-(tert-butyl)acetoacetamide                                                                      2-(tert-butylcarbamoyl)-1-methyl-                                             ethyl-BM123γ      0.38                              1,1-dimethoxyacetone                                                                              1-methyl-2,2-dimethoxyethyl-BM123γ                                                              0.5                               4-heptanone         1-propylbutyl-BM123γ                                                                            0.25                              3-methoxyphenylacetone                                                                            1-methyl-2-(3-methoxyphenyl)ethyl-                                            BM123γ            0.30                              1,3-acetonedicarboxylic acid                                                                      (2-carboxy-1-carboxymethyl)ethyl-                                             BM123γ            0.5                               2-phenylcyclohexanone                                                                             2-phenylcyclohexyl-BM123γ                                                                       0.38                              phenoxy-2-propanone 1-methyl-2-phenoxyethyl-BM123γ                                                                  0.3                               3-butyn-2-one       1-methyl-butyn(-2-)yl-BM123γ                                                                    <2.0                              dimethylaminoacetone                                                                              1-methyl-2-(N,N-dimethylamino)propyl-                                         BM123γ            1.5                               5-diethylamino-2-pentanone                                                                        1-methyl-4-(N,N-diethylamino)butyl-                                           BM123γ            0.5                               2-cyclohexen-1-one  2-cyclohexenyl-BM123γ                                                                           0.25                              cyclopropylmethylketone                                                                           1-cyclopropylethyl-BM123γ                                                                       0.25                              4,4-dimethoxy-2-butanone                                                                          1-methyl-3,3-dimethoxypropyl-BM123γ                                                             0.75                              1,3-dimethylacetonedicarboxylate                                                                  1-carbomethoxy-2-carbomethoxyethyl-                                           BM123γ            0.75                              2-methoxyphenylacetone                                                                            1-methyl-2-(2-methoxyphenyl)ethyl-                                                                    0.7                                                   BM123γ                                              acetylacetone       1-methyl-2-acetylethyl-BM123γ                                                                   1.5                               cyclobutanone       cyclobutyl-BM123γ 0.38                              p-chlorophenylacetone                                                                             1-methyl-2-(4-chlorophenyl)ethyl-                                             BM123γ            0.25                              2-octanone          1-methylheptyl-BM123γ                                                                           0.38                              4-phenyl-2-butanone 1-methyl-3-phenylpropyl-BM123γ                                                                  0.38                              5-chloro-2-pentanone                                                                              1-methyl-4-chlorobutyl-BM123γ                                                                   0.37                              o-chlorophenylacetone                                                                             1-methyl-2-(2-chlorophenyl)ethyl-                                             BM123γ            0.37                              m-chlorophenylacetone                                                                             1-methyl-2-(3-chlorophenyl)ethyl-                                             BM123γ            0.38                              5-hexene-2-one      1-methyl-penten(-4-)yl-BM123γ                                                                   0.38                              cyclohexanone       cyclohexyl-BM123γ 0.75                              2-hexanone          1-methylpentyl-BM123γ                                                                           0.38                              2-heptanone         1-methylhexyl-BM123γ                                                                            0.38                              cycloheptanone      cycloheptyl-BM123γ                                                                              0.3                               cyclopentanone      cyclopentyl-BM123γ                                                                              0.25                              4,4-dimethyl-2-pentanone                                                                          1,3,3-trimethylbutyl-BM123γ                                                                     0.18                              2-acetamido-3-butanone                                                                            2-acetamido-1-methylpropyl-BM123γ                                                               0.50                              2,6-dimethyl-3-heptanone                                                                          1-isopropyl-4-methylpentyl-BM123γ                                                               0.39                              4-octanone          1-propylpentyl-BM123γ                                                                           0.39                              3-acetylpyridine    1-(3-pyridyl)ethyl-BM123γ                                                                       0.75                              3-heptanone         1-ethylpentyl-BM123γ                                                                            0.25                              ethyl butyrylacetate                                                                              1-(carbethoxymethyl)butyl-BM123γ                                                                0.75                              1-benzyl-4-piperidone                                                                             1-benzyl-4-piperidyl-BM123γ                                                                     0.18                              1-methyl-4-piperidone                                                                             1-methyl-4-piperidyl-BM123γ                                                                     0.75                              3-methylcyclopentanone                                                                            3-methylcyclopentyl-BM123γ                                                                      0.25                              3,3-dimethyl-2-butanone                                                                           1-methyl-2,2-dimethylpropyl-BM123γ                                                              0.18                              2-acetyl-5-norbornene                                                                             1-[5-norbornene(2)]ethyl-BM123γ                                                                 <0.12                             bicyclo[3.2.1]octan-2-one                                                                         bicyclo[3.2.1]octanyl-2-BM123γ                                                                  <0.25                             3-quinuclidinone    3-quinuclidinyl-BM123γ                                                                          0.39                              5-methoxyl-2-tetralone                                                                            5-methoxyl-2-tetralyl-BM123γ                                                                    0.18                              4-methyl-2-heptanone                                                                              1,3-dimethylhexyl-BM123γ                                                                        0.5                               3,4-dimethyl-2-hexanone                                                                           1,2,3-trimethylpentyl-BM123γ                                                                    0.39                              1,3,3-trimethylcyclopentanone                                                                     1,3,3-trimethylcyclopentyl-BM123γ                                                               0.37                              acetylcyclopentanone                                                                              1-cyclopentylethyl-BM123γ                                                                       0.18                              5-hexen-2-one       1-methyl-hexen-4-yl-BM123γ                                                                      0.18                              2-methylcyclopentanone                                                                            2-methylcyclopentyl-BM123γ                                                                      0.25                              2,4-dimethylcyclopentanone                                                                        2,4-dimethylcyclopentyl-BM123γ                                                                  0.18                              2-ethylcyclopentanone                                                                             2-ethylcyclopentyl-BM123γ                                                                       0.12                              2-adamantone        adamantyl-2-BM123γ                                                                              0.25                              3-hexanone          1-ethylbutyl-BM123γ                                                                             0.39                              ethyl 2-methylacetoacetate                                                                        1-methyl-2-carboethoxypropyl-BM123γ                                                             0.18                              norbornanone        norbornyl-BM123γ  0.18                              5-hexene-2-one      1-methyl-5-pentenyl-BM123γ                                                                      0.18                              3-hydroxy-2-butenone                                                                              1-methyl-2-hydroxypropyl-BM123γ                                                                 0.37                              4-hydroxy-3-methyl-2-butanone                                                                     1-methyl-2-(3-hydroxy-2-methylpropyl)-                                        BM123γ            0.18                              2-nonanone          1-methyloctyl-BM123γ                                                                            0.37                              5-hydroxy-2-pentanone                                                                             1-methyl-4-hydroxybutyl-BM123γ                                                                  0.18                              2-decanone          1-methylnonyl-BM123γ                                                                            0.18                              4-t-butylcyclohexanone                                                                            4-t-butylcyclohexyl-BM123γ                                                                      0.12                              2-ethylidenecyclohexanone                                                                         2-ethylidenecyclohexyl-BM123γ                                                                   0.12                              phenylacetaldehyde  2-phenylethyl-BM123γ                                                                            0.18                              p-methoxyphenylacetaldehyde                                                                       2-(p-methoxyphenyl)ethyl-BM123γ                                                                 0.18                              2-ethylhexanal      2-ethylhexyl-BM123γ                                                                             0.37                              2,2-dimethylbutanal 2,2-dimethylbutyl-BM123γ                                                                        0.12                              2,2-dimethylpropanal                                                                              2,2-dimethylpropyl-BM123γ                                                                       0.18                              2-ethyl-2-butenal   2-ethyl-2-butenyl-BM123γ                                                                        0.18                              trans-2-methyl-2-butenal                                                                          trans-2-methyl-2-butenyl-BM123γ                                                                 0.18                              1-methylcyclo-3-hexenylmethanal                                                                   (1-methylcyclo-3-hexenyl)methyl-BM123γ                                                          0.18                              trans-2-methyl-2-pentenal                                                                         trans-2-methyl-2-pentenyl-BM123γ                                                                0.18                              formaldehyde        methyl-BM123γ     0.12                              acetaldehyde        ethyl-BM123γ      0.38                              __________________________________________________________________________

Fermentation Process Selected to Produce Primarily BM123β and BM123γ.

Cultivation of Nocardia sp. NRRL 8050 may be carried out in a widevariety of liquid culture media. Media which are useful for theproduction of the antibiotics include an assimilable source of carbonsuch as starch, sugar, molasses, glycerol, etc.; an assimilable sourceof nitrogen such as protein, protein hydrolyzate, polypeptides, aminoacids, corn steep liquor, etc.; and inorganic anions and cations, suchas potassium, magnesium, calcium, ammonium, sulfate, carbonate,phosphate, chloride, etc. Trace elements such as boron, molybdenum,copper, etc.; are supplied as impurities of other constituents of themedia. Aeration in tanks and bottles is provided by forcing sterile airthrough or onto the surface of the fermenting medium. Further agitationin tanks is provided by a mechanical impeller. An antifoaming agent,such as Hodag® FD82 may be added as needed.

Inoculum Preparation for BM123β and BM123γ

Primary shaker flask inoculum of Nocardia sp. NRRL 8050 is prepared byinoculating 100 milliliters of sterile liquid medium in 500 milliliterflasks with scrapings or washings of spores from an agar slant of theculture. The following medium is ordinarily used:

    ______________________________________                                        Bacto-tryptone      5       gm.                                               Yeast extract       5       gm.                                               Beef extract        3       gm.                                               Glucose             10      gm.                                               Water to            1000    ml.                                               ______________________________________                                    

The flasks were incubated at a temperature from 25°- 29° C., preferably28° C. and agitated vigorously on a rotary shaker for 30 to 48 hours.The inocula are then transferred into sterile screw cap culture tubesand stored at below 0° F. This bank of vegetative inoculum is usedinstead of slant scrapings for inoculation of additional shaker flasksin preparation of this first stage of inoculum.

These first stage flask inocula are used to seed 12 liter batches of thesame medium in 20 liter glass fermentors. The inoculum mash is aeratedwith sterile air while growth is continued for 30 to 48 hours.

The 12 liter batches of second stage inocula are used to seed tankfermentors containing 300 liters of the following sterile liquid mediumto produce the thrid and final stage of inoculum:

    ______________________________________                                        Meat solubles            15      gm.                                          Ammonium sulfate         3       gm.                                          Potassium phosphate, dibasic                                                                           3       gm.                                          Calcium carbonate        1       gm.                                          Magnesium sulfate heptahydrate                                                                         1.5     gm.                                          Glucose                  10      gm.                                          Water to                 1000    ml.                                          The glucose is sterilized separately.                                         ______________________________________                                    

The third stage inoculum is aerated at 0.4 to 0.8 liters of sterile airper liter of broth per minute, and the fermenting mixture is agitated byan impeller driven at 150-300 revolutions per minute. The temperature ismaintained at 25°-29° C., usually 28° C. The growth is continued for 48to 72 hours, at which time the inoculum is used to seed a 3000 litertank fermentation.

Tank Fermentation for BM123β and BM123γ

For the production of BM123β and BM123γ in tank fermentors, thefollowing fermentation medium is preferably used:

    ______________________________________                                        Meat solubles            30      gm.                                          Ammonium sulfate         6       gm.                                          Potassium phosphate, dibasic                                                                           6       gm.                                          Calcium carbonate        2       gm.                                          Magnesium sulfate heptahydrate                                                                         3       gm.                                          Glucose                  20      gm.                                          Water to                 1000    ml.                                          The glucose is sterilized separately.                                         ______________________________________                                    

Each tank is inoculated with 5 to 10% of third stage inoculum made asdescribed under inoculum preparation. The fermenting mash is maintainedat a temperature of 25°-28° C. usually 26° C. The mash is aerated withsterile air at a rate of 0.3-0.5 liters of sterile air per liter of mashper minute and agitated by an impeller driven at 70 to 100 revolutionsper minute. The fermentation is allowed to continue from 65-90 hours andthe mash is harvested.

The invention will be described in greater detail in conjunction withthe following specific examples.

EXAMPLE 1 Inoculum preparation for BM123β and BM123γ

A typical medium used to grow the first and second stages of inoculumwas prepared according to the following formula:

    ______________________________________                                        Bacto-tryptone      5       gm.                                               Yeast extract       5       gm.                                               Beef extract        3       gm.                                               Glucose             10      gm.                                               Water to            1000    ml.                                               ______________________________________                                    

Two 500 milliliter flasks each containing 100 milliliters of the abovesterile medium were inoculated with 5 milliliters each of a frozenvegetative inoculum from Nocardia sp. NRRL 8050. The flasks were placedon a rotary shaker and agitated vigorously for 48 hours at 28° C. Theresulting flask inoculum was transferred to a 5 gallon glass fermentorcontaining 12 liters of the above sterile medium. The mash was aeratedwith sterile air while growth was carried out for about 48 hours, afterwhich the contents were used to seed a 100 gallon tank fermentorcontaining 300 liters of the following sterile liquid medium:

    ______________________________________                                        Meat solubles            15      gm.                                          Ammonium sulfate         3       gm.                                          Potassium phosphate, dibasic                                                                           3       gm.                                          Calcium carbonate        1       gm.                                          Magnesium sulfate heptahydrate                                                                         1.5     gm.                                          Glucose                  10      gm.                                          Water to                 1000    ml.                                          The glucose is sterilized separately.                                         ______________________________________                                    

The third stage of inoculum mash was aerated with sterile air spargedinto the fermentor at 0.4 liters of air per liter of mash per minute.Agitation was supplied by a driven impeller at 240 revolutions perminute. The mash was maintained at 28° C. and Hodag® FD82 was used as adefoaming agent. After 48 hours of growing time the inoculum mash wasused to seed a 3000 liter fermentation.

EXAMPLE 2 Fermentation Employing Nocardia sp. NRRL 8050 and MediumFavoring the Production of BM123β and BM123γ

A fermentation medium was prepared according to the following formula:

    ______________________________________                                        Meat solubles            30      gm.                                          Ammonium sulfate         6       gm.                                          Potassium phosphate, dibasic                                                                           6       gm.                                          Calcium carbonate        2       gm.                                          Magnesium sulfate heptahydrate                                                                         3       gm.                                          Glucose                  20      gm.                                          Water to                 1000    ml.                                          The glucose is sterilized separately.                                         ______________________________________                                    

The fermentation medium was sterilized at 120° C. with steam at 20pounds pressure for 60 minutes. The pH of the medium after sterilizationwas 6.9. Three thousand liters of sterile medium in a 4000 liter tankfermentor was inoculated with 300 liters of inoculum such as describedin Example 1, and the fermentation was carried out at 26° C. usingHodag® FD82 as a defoaming agent. Aeration was supplied at the rate of0.35 liter of sterile air per liter of mash per minute. The mash wasagitated by an impeller driven at 70-72 revolutions per minute. At theend of 67 hours of fermentation time the mash was harvested.

EXAMPLE 3 Isolation of BM123β and BM128γ

A 3000 liter portion of fermentation mash prepared as described inExample 2, pH 4.3, was adjusted to pH 7.0 with sodium hydroxide andfiltered using 5% diatomaceous earth as a filter aid. The cake waswashed with about 100 liters of water and discarded. The combinedfiltrate and wash was pumped upward through three parallel 81/4 inches ×48 inches stainless steel columns each containing 15 liters of CMSephadex® C-25 [Na⁺ ] resin (a cross-linked dextran-epichlorohydrincation exchange gel available from Pharmacia Fine Chemicals, Inc.). Thecharged columns were washed with a total of about 390 liters of waterand then developed with 200 liters of 1% aqueous sodium chloridefollowed by 560 liters of 5% aqueous sodium chloride. The 5% aqueoussodium chloride eluate was clarified by filtration through diatomaceousearth and the clarified filtrate passed through a 9 inches × 60 inchesglass column containing 25 liters off granular Darco® G-60 (20-40 mesh)(a granular activated carbon available from Atlas Chemical Industries,Inc.). The charged column was washed with 120 liters of water and thendeveloped with 120 liters of 15% aqueous methanol followed by 340 litersof 50% aqueous methanol and then 120 liters of 50% aqueous acetone. The15% aqueous methanol eluate was concentrated in vacuo to about 7 litersof an aqueous phase and the pH adjusted from 4.5 to 6.0 with Amberlite®IR-45 (OH⁻) resin (a weakly basic polystyrene-polyamine type anionexchange resin). The resin was removed by filtration and the filtratewas concentrated in vacuo to about 1 liter and then lyophilized to give38 grams of material consisting primarily of BM123β along with a smallamount of BM123γ (primarily BM123γ₂). The 50% aqueous methanol eluatewas adjusted from pH 4.65 to 6.0 with Amberlite® IR-45 (OH⁻) resin. Theresin was removed by filtration and the filtrate was concentrated invacuo to about 6.3 liters and then lyophylized to give 213 grams ofmaterial consisting primarily of BM123γ. The 50% aqueous acetone eluatewas adjusted from pH 4.0 to 6.0 with Amberlite® IR-45 (OH⁻) resin. Theresin was removed by filtration and the filtrate was concentrated invacuo to about 1.5 liters and then lyophylized to give 56 grams ofimpure BM123γ.

EXAMPLE 4 Further Purification of BM123γ

A slurry of CM Sephadex® C-25 [NH₄ ⁺ ] in 2% aqueous ammonium chloridewas poured into a 2.6 centimeter diameter glass column to a resin heightof approximately 62 centimeters. The excess 2% aqueous ammonium chloridewas drained away and a 5.0 gram sample of BM123γ prepared as describedin Example 3 was dissolved in about 10 milliliters of 2% aqueousammonium chloride and applied to the column. The column was then elutedwith a gradient between 6 liters each of 2% and 4% aqueous ammoniumchloride. Fractions of about 75 milliliters each were collectedautomatically every 15 minutes. Antibiotic BM123γ was located bymonitoring the column effluent in the ultraviolet and by bioautographyof dipped paper disks on large agar plates seeded with Klebsiellapneumoniae strain AD. The majority of BM123γ was located betweenfractions 71-107 inclusive.

One hundred thirty milliliters of granular Darco® G-60 (2040 mesh) wassuspended in water, transferred to a glass column, allowed to settle andthe excess water was allowed to drain away. Fractions 84-96 inclusivefrom the above CM Sephadex chromatography were combined and passedthrough the granular carbon column. The charged column was washed with600 milliliters of water and then developed with 1 liter of 50% aqueousacetone. The eluates, both of which contained BM123β, were concentratedto aqueous phases in vacuo and lyophilized to give a total of 886milligrams of BM123γ as the hydrochloride salt. A microanalytical samplewas obtained by subjecting the above material to a repeat of the aboveprocess.

Antibiotic BM123γ does not possess a definite melting point, but gradualdecomposition starts in the vicinity of 200° C. Microanalysis of asample equilibrated for 25 hours in a 72° F. atmosphere containing 23%relative humidity gave C, 39.44%; H, 6.10%; N, 16.19%; Cl(ionic),11.54%; loss on drying, 8.19%. In water BM123γ gave a U.V. absorptionmaximum at 286 nm with E_(1cm) ^(1%) = 250. The position of this maximumdid not change with pH. BM123γ had a specific rotation of [α]_(D) ²⁵°+71° (C = 0.97 in water).

Antibiotic BM123γ exhibited characteristic absorption in the infraredregion of the spectrum at the following wavelengths: 770, 830, 870, 930,980, 1035, 1105, 1175, 1225, 1300, 1340, 1370, 1460, 1510, 1555, 1605,1660, 1740, 2950 and 3350 cm⁻ 1. A standard infrared absorption spectrumof BM123γ prepared in a KBr pellet is shown in FIG. 1 of theaccompanying drawings.

EXAMPLE 5 Isolation of BM123γ₁

A slurry of CM Sephadex® C-25 [Na⁺ ] in 2% aqueous sodium chloride waspoured into a 2.6 centimeter diameter glass column to a resin heigh ofapproximately 70 centimeters. The excess 2% aqueous sodium chloride wasdrained away and 4.11 gram of a sample containing primarily BM123γ₁along with some BM123γ₂ and other impurities, prepared as described inExample 3, was dissolved in about 10 milliliters of 2% aqueous sodiumchloride and applied to the column. The column was then eluted with agradient between 4 liters each of 2% and 4% aqueous sodium chloride.Fractions of about 75 milliliters each were collected automaticallyevery 15 minutes. Antibiotic BM123γ was located by monitoring the columneffluent in the ultraviolet and by bioautography of dipped paper diskson large agar plates seeded with Klebsiella pneumoniae strain AD. Themajority of BM123γ was located between fractions 64-90 inclusive; theinitial fractions (64-80) contained a mixture of BM123γ₁ and BM123γ₂whereas the later fractions (81-90) contained essentially pure BM123γ₁.

One hundred milliliters of granular Darco® G-60 (20-40 mesh) wassuspended in water, transferred to a glass columm, allowed to settle andthe excess water was allowed to drain away. Fractions 81-90 inclusivefrom the above CM Sephadex chromatography were combined and passedthrough the granular carbon column. The charged column was washed with500 milliliters of water and then developed with 500 milliliters of 10%aqueous methanol followed by 1 liter of 50% aqueous methanol. The 50%aqueous methanol eluate, which contained the majority of BM123γ₁, wasadjusted from pH 5.9 to 6.0 with Amberlite® IR-45(OH⁻¹) resin. The resinwas removed by filtration and the filtrate was concentrated in vacuo toan aqueous phase and lyophilized to give 294 milligrams of whiteamorphous BM123γ₁ as the hydrochloride salt.

Antibiotic BM123γ₁ does not possess a definite melting point, butgradual decomposition starts in the vicinity of 200° C. Microanalysis ofa sample equilibrated for 24 hours in a 70° F. atmosphere containing 60%relative humidity gave C, 37.84%; H, 5.73%; N, 15.58%; Cl(ionic),10.01%, loss on drying 10.45%. In methanol BM123γ₁ gave a U.V.absorption maximum at 286 nm with E_(1cm) ^(1%) = 225. The position ofthis maximum did not change with pH. BM123γ₁ had a specific rotation of+55° (C=0.803 in water).

Antibiotic BM123γ₁ exhibited characteristic absorption in the infraredregion of the spectrum at the following wavelengths: 770, 830, 870, 930,980, 1045, 1080, 1110, 1125, 1175, 1225, 1305, 1345, 1380, 1465, 1515,1560, 1605, 1660, 1730, 2950 and 3350 cm⁻¹. A standard infraredabsorption spectrum of BM123γ₁ prepared in a KBr pellet is shown in FIG.2 of the accompanying drawings. A standard proton magnetic resonancespectrum of BM123γ₁ determined on a D₂ O solution in a 100 megacyclespectrometer is shown in FIG. 4 of the accompanying drawings.

EXAMPLE 6 Isolation of BM123γ₂

A 25 gram sample containing primarily BM123γ₂ and BM123β, prepared asdescribed in Example 3, was dissolved in about 120 milliliters of 2%aqueous sodium chloride and applied to a column containing 1800 ml. ofCM Sephadex® C-25 [Na⁺ ] in 2% aqueous sodium chloride. The column wasthen eluted with a gradient between 20 liters each of 2% and 4% aqueoussodium chloride. The initial 12 liters of eluate was collected in alarge bottle and discarded. Thereafter fractions of about 800milliliters each were collected automatically every 40 minutes.Antiobiotic BM123γ was located by monitoring the column fractions in theultraviolet. The majority of BM123γ was located between fractions 7-19inclusive; the initial fractions (7-15) contained essentially pureBM123γ₂ and the later fractions (16-18) contained a mixture of BM123γ₁and BM123γ₂.

Six hundred milliliters of granular Darco® G-60 (20-40 mesh) wassuspended in water, transferred to a glass column, allowed to settle andthe excess water was allowed to drain away. Fractions 7-15 inclusivefrom the above CM Sephadex chromatography were combined and passedthrough the granular carbon column. The charged column was washed with 3liters of water and then developed with 3 liters of 10% aqueous methanolfollowed by 6 liters of 50% aqueous methanol. The 10% aqueous methanoleluate was adjusted from pH 5.8 to 6.0 with Amberlite® IR 45 (OH⁻)resin. The resin was removed by filtration and the filtrate wasconcentrated in vacuo to an aqueous phase and lyophilized to give 595milligrams of white amorphous BM123γ₂ as the hydrochloride salt. The 50%aqueous methanol eluate was adjusted from pH 4.6 to 6.1 with Amberlite®IR 45 (OH⁻) resin. The resin was removed by filtration and the filtratewas concentrated in vacuo to an aqueous phase and lyophilized to give3.645 grams of slightly less pure white amorphous BM123γ₂ as thehydrochloride salt.

Antibiotic BM123γ₂ does not possess a definite melting point, butgradual decomposition starts in the vicinity of 200° C. Microanalysis ofa sample equilibrated for 24 hours in a 70° F. atmosphere containing 60%relative humidity gave C, 36.14%; H, 5.67%, N, 15.1%; Cl(ionic), 11.11%;loss on drying 10.87%. In methanol BM123γ₂ gave a U.V. absorptionmaximum at 286 nm with E_(1cm) ^(1%) = 220. The position of this maximumdid not change with pH. BM123γ₂ had a specific rotation of +60° (C=0.851in water).

Antibiotic BM123γ₂ exhibited characteristic absorption in the infraredregion of the spectrum at the following wavelengths: 770, 830, 870, 950,1035, 1110, 1175, 1225, 1285, 1345, 1380, 1470, 1515, 1560, 1605, 1660,1755, 2950 and 3350 cm⁻¹. A standard infrared absorption spectrum ofBM123γ₂ prepared in a KBr pellet is shown in FIG. 3 of the accompanyingdrawings. A standard proton magnetic resonance spectrum of BM123γ₂determined on a D₂ O solution in a 100 megacycle spectrometer is shownin FIG. 5 of the accompanying drawings.

EXAMPLE 7 Paper Partition and Thin Layer Chromatography of BM123β andBM123γ

the BM123 antibiotics can be distinguished by paper chromatography. Forthis purpose Whatman No. 1 strips were spotted with a water or methanolsolution of the substances and equilibrated for 1 to 2 hours in thepresence of both upper and lower phases. The strips were developedovernight with the lower (organic) phase obtained from mixing 90%phenol:m-cresol:acetic acid:pyridine:water (100:25:4:4:75 by volume).The developed strips were removed from the chromatographic chamber, airdried for 1 to 2 hours, washed with ether to remove residual phenol andbioautographed on large agar plates seeded with Klebsiella peunmoniaestrain AD. Representative Rf values are listed in Table VII below:

                  TABLE VII                                                       ______________________________________                                        Component        RF                                                           ______________________________________                                        BM123γ     0.85                                                         BM123β      0.50, 0.70                                                   ______________________________________                                    

The β component was a mixture of two antibiotics using this system.BM123β was composed of a major antibiotic (Rf = 0.50) called BM123β₁ anda minor antibiotic (Rf = 0.70) called BM123β₂.

The BM123 antibiotics can also be distinguished by thin layerchromatography. For this purpose pre-coated Cellulose F® plates (0.10millimeters thick), a form of thick layer cellulose supplied by EMLaboratories Inc., Elmsford, N.Y. were spotted with a water solution ofthe substance to be chromatographed (about 20-40 micrograms per spot).The plates were developed overnight with the solvent obtained by mixing1-butanol:water:pyridine:acetic acid (15:12:10:1 by volume). Thedeveloped plates were removed from the chromatographic chamber and airdried for about 1 hour. The antibiotics were detected by using eitherstandard ninhydrin or Sakaguchi spray reagents. Representative Rf valuesare listed in Table VIII below:

                  TABLE VIII                                                      ______________________________________                                        Component        Rf                                                           ______________________________________                                        BM123γ     0.17, 0.23                                                   BM123β      0.08, 0.14                                                   ______________________________________                                    

Both BM123β and γ were a mixture of two components using this system.BM123β was composed of a major component (Rf = 0.08) which was BM123β₁and a minor component (Rf = 0.14) which was BM123β₂. The less polarcomponent of BM123γ (Rf = 0.23) was BM123γ₁ and the more polar component(Rf = 0.17) was BM123γ₂.

EXAMPLE 8 General Procedure for Reductive Alkylation of AntibioticBM123γ

To a stirred solution of 100 mg. of antibiotic BM123γ in 20 ml. ofmethanol is added 5 ml. (or 5 g.) of the appropriate aldehyde or ketoneand 100 mg. of sodium cyanoborohydride. The pH of the resulting solutionis maintained at about 7.0 with 0.1N methanolic hydrogen chloride over a3 to 24 hour period. The reaction is monitored by thin layerchromatography to the disappearance of the BM123γ. The reaction mixtureis then filtered and the filtrate is evaporated to dryness. The residueis triturated with 3 ml. of methanol and filtered. The filtrate isdiluted with 50 ml. of acetone and the precipitate which forms isremoved by filtration and dried. The methanol solvent may be replaced by20 ml. of water wherever the starting aldehyde or ketone is watersoluble.

EXAMPLE 9 Preparation of methyl-BM123γ

To a solution of 1.0 g. of BM123γ and 2.5 ml. of a 37% aqueousformaldehyde solution in 50 ml. of water was added, portionwise, 400 mg.of sodium cyanoborohydride. The pH of the reaction mixture wasmaintained at 7.0 with 1N hydrochloric acid during this addition. Thereaction mixture was stirred an additional ten minutes at roomtemperature and then evaporated to dryness to vacuo. The residue wastriturated with 20 ml. of methanol, filtered and the filtrate dilutedwith 250 ml. of acetone. The product which precipitated was removed byfiltration and dried; yield, 667 mg.

EXAMPLE 10 Preparation of isopropyl-BM123γ

To a solution of 200 mg. of BM123γ in 30 ml. of methanol was added 5 ml.of acetone. To this solution was added 139 mg. of sodiumcyanoborohydride and the reaction mixture was stirred at roomtemperature for 30 minutes. During this time the pH of the reactionmixture was maintained between 7.4 and 7.8 by the addition of 0.1Nmethanolic hydrogen chloride. The small amount of precipitate which hadformed was removed by filtration and the filtrate was evaporated todryness in vacuo. The residue was triturated with two ml. of methanoland filtered. The filtrate was diluted with 100 ml. of acetone and thesolid product that separated was removed by filtration and dried; yield,184 mg.

EXAMPLE 11 Preparation of β-phenylethyl-BM123γ

To a solution of 200 mg of BM123γ in 15 ml. of water and 25 ml. ofacetonitrile was added a solution of 2 ml. of phenylacetaldehyde in 4ml. of ethanol. To this was added 103 mg. of sodium cyanoborohydride.The reaction mixture was stirred at room temperature for 30 minutesduring which time the pH of the mixture was maintained at 7 with 0.2Nhydrochloric acid. The reaction mixture was then filtered and thefiltrate was evaporated to dryness in vacuo. The residue was trituratedwith two ml. of methanol and filtered. The filtrate was diluted with 100ml. of acetone and the product that separated was removed by filtrationand dried; yield, 180 mg.

EXAMPLE 12 Preparation of 1,3,3-trimethylbutyl-BM123γ

To a solution of 200 mg. of BM123γ hydrochloride in 50 ml. of methanolwas added 3 ml. of 4,4-dimethyl-2-pentanone and 106 mg. of sodiumcyanoborohydride. The reaction solution was maintained at pH 7 by thedropwise addition of methanolic hydrogen chloride. The reaction wasstirred at room temperature for 18 hours and filtered. The filtrate wasevaporated to dryness in vacuo. The residue was dissolved in 3 ml. ofmethanol, diluted with 50 ml. of acetone and filtered, yield 125 mg.

EXAMPLE 13 Preparation of 1-methylphenethyl-BM123γ

To a solution of 200 mg. of BM123γ in 50 ml. of methanol was added 5 ml.of phenylacetone. To this solution was added 170 mg. of sodiumcyanoborohydride and the reaction mixture stirred at room temperaturefor 3 and a half hours. During this time the pH of the reaction mixturewas maintained at 7.0 with methanol saturated with hydrogen chloridegas. Reaction mixture was concentrated to about 5 ml. volume, dilutedwith two ml. of methanol, and filtered. Filtrate was poured into 100 ml.of acetone and the solid product that separated was removed byfiltration and dried; yield, 233 mg.

EXAMPLE 14 Preparation of 1-methylnonyl-BM123γ

Sodium cyanoborohydride (100 mg.) was added to a solution of BM123γ (200mg.) and 2-decanone (1 ml.) in 40 ml. of methanol. The pH of thesolution was adjusted to 7.0 and maintained at 7.0 ± 0.2 by the additionof 0.1N methanolic hydrogen chloride as necessary. After 19.5 hours thereaction mixture was filtered and the filtrate was concentrated in vacuoat 35° C. The residue was slurried in 5 ml. of methanol and filtered.The filtrate was added to 50 ml. of acetone. The off white solid whichprecipitated was collected by filtration, washed with acetone, and driedin vacuo. The yield of crude 1-methylnonyl-BM123γ was 167 mg.

EXAMPLE 15 Preparation of 1,3-dimethylbutyl-BM123γ

To a solution of 210 mg. of BM123γ in 50 ml. of methanol was added 5 ml.of methyl isobutyl ketone. To this solution was added 166 mg. of sodiumcyanoborohydride and the reaction mixture strired at room temperaturefor five hours. During this time the pH of the reaction mixture wasmaintained at 7.0 with methanol saturated with hydrogen chloride gas.Reaction mixture was evaporated to dryness, in vacuo. The residue wastriturated with two ml. of methanol and filtered. The filtrate wasdiluted with 100 ml. of acetone and the solid product that separated wasremoved by filtration and dried; yield, 210 mg.

EXAMPLE 16 Preparation of 2-norbornyl-BM123γ

Sodium cyanoborohydride (100 mg.) was added to a solution of BM123γ (200mg.) and 2-norbornanone (400 mg.) in 40 ml. of methanol. The pH of thesolution was adjusted to 7.0 with 0.1N methanolic hydrogen chloride. ThepH was maintained at 7.0 ± 0.2 by the addition of 0.1N hydrogen chlorideas necessary. After 21.5 hours the reaction mixture was filtered and thefiltrate was concentrated in vacuo at 35° C. The residue was slurried in5 ml of methanol and filtered. The filtrate was added to 50 ml. ofacetone. The off white solid which precipitated was collected byfiltration, washed with acetone and dried in vacuo. The yield of crude2-norbornyl-BM123γ was 175 mg.

EXAMPLE 17 Preparation of isopropyl-BM123γ₁

A mixture of 50 mg. of BM123γ₁, 5 ml. of acetone and 60 mg. of sodiumcyanoborohydride in 35 ml. of methanol was stirred at room temperaturefor 40 minutes. The pH of the solution was maintained at 7 by thedropwise addition of a methanolic hydrogen chloride solution. Themixture was evaporated to dryness in vacuo. The residue was trituratedwith 5 ml. of methanol and the resulting solution was diluted with 50ml. of acetone; yield, 49 mg.

EXAMPLE 18 Preparation of isopropyl-BM123γ₂

A mixture of 41 mg. of BM123γ₂, 5 ml of acetone and 50 mg. of sodiumcyanoborohydride in 35 ml. of methanol was stirred at room temperaturefor 40 minutes. The pH of the solution was maintained at 7 by thedropwise addition of a methanolic hydrogen chloride solution(saturated). The mixture was filtered and evaporated to dryness invacuo. The residue was triturated with 5 ml. of methanol and theresulting solution was diluted with 50 ml. of acetone; yield, 46 mg.

EXAMPLE 19 Preparation of 1-methyl-2-phenyl-ethyl-BM123γ₂

A mixture of 200 mg. of BM123γ₂, 5 ml of phenylacetone and 170 mg. ofsodium cyanoborohydride in 50 ml. of methanol was stirred at roomtemperature for 3 hours and 45 minutes. During this time the pH of thereaction mixture was maintained at 7 with dropwise addition of amethanolic hydrogen chloride solution (saturated). The mixture wasevaporated to dryness in vacuo. The residue was triturated with 5 ml. ofmethanol and the resulting methanol solution was diluted withapproximately 50 ml. of acetone; yield 233 mg.

EXAMPLE 20 Preparation of (2-ethylcyclopentyl) BM123γ

A solution of 200 mg. of BM123γ, 3 ml. of 2-ethylcyclopentanone and 101mg. of sodium cyanoborohydride in 50 ml. of methyl alcohol was stored atroom temperature for 18 hours. During this time the pH of the solutionwas maintained at 7 with the addition of a saturated solution ofhydrogen chloride in methanol. The reaction mixture was evaporated todryness. The residue was triturated with 3 ml. of methanol, filtered andthe filtrate was diluted with 40 ml. of acetone, yield, 157 mg.

EXAMPLE 21 Preparation of 3,5-dimethylcyclohexyl BM123γ

A solution of 200 mg. of BM123γ, 5 ml. of 3,5-dimethylcyclohexanone and200 mg. of sodium cyanoborohydride in 50 ml. of methanol was stored atroom temperature for 1 hour. During this time the pH of the solution wasmaintained at 7 with the addition of a saturated solution of hydrogenchloride in methanol. The reaction was triturated with 3 ml. ofmethanol, filtered and the filtrate was diluted with 40 ml. of acetone,yield 200 mg.

EXAMPLE 22 Preparation of 2,4-dimethylcyclopentyl BM123γ

A solution of 206 mg. of BM123γ, 3 ml. of 2,4-dimethylcyclopentanone and104 mg. of sodium cyanoborohydride in 50 ml. of methanol was stored atroom temperature for 6 hours. During this time the pH of the solutionwas maintained at 7 with the addition of a saturated solution ofhydrogen chloride in methanol. The reaction was triturated with 3 ml. ofmethanol, filtered and the filtrate was diluted with 40 ml. of acetone,yield 101 mg.

EXAMPLE 23 Preparation of 2-ethylcyclohexyl BM123γ

A solution of 200 mg. of BM123γ, 5 of 2-ethylcyclohexanone and 213 mg.of sodium cyanoborohydride in 50 ml. of methanol was stored at roomtemperature for 3 hours. During this time the pH of the solution wasmaintained at 7 with the addition of a saturated solution of hydrogenchloride in methanol. The reaction was triturated with 3 ml. ofmethanol, filtered and the filtrate was diluted with 40 ml. of acetone,yield 200 mg.

EXAMPLE 24 Preparation of 3-methylcyclohexyl BM123γ

A solution of 200 mg. of BM123γ, 1.5 ml. of 3-methylcyclohexanone and200 mg. of sodium cyanoborohydride in 50 ml. of methanol was stored atroom temperature for 2 hours. During this time the pH of the solutionwas maintained at 7 with the addition of a saturated solution ofhydrogen chloride in methanol. The reaction was triturated with 3 ml. ofmethanol, filtered and the filtrate was diluted with 40 ml. of acetone,yield 200 mg.

EXAMPLE 25 Preparation of 2,4,4-trimethylcyclopentyl BM123γ

A solution of 200 mg. of BM123γ, 5 ml. of 2,4,4-trimethylcyclopentanoneand 179 mg. of sodium cyanoborohydride in 50 ml. of methanol was storedat room temperature for 24 hours. During this time the pH of thesolution was maintained at 7 with the addition of a saturated solutionof hydrogen chloride in methanol. The reaction was triturated with 3 ml.of methanol, filtered and the filtrate was diluted with 40 ml. ofacetone, yield 176 mg.

EXAMPLE 26 Preparation of 2-propylcyclohexyl BM123γ

A solution of 200 mg. of BM123γ, 3 ml. of 2-propylcyclohexanone and 157mg. of sodium cyanoborohydride in 50 ml. of methanol was stored at roomtemperature for 4 hours. During this time the pH of the solution wasmaintained at 7 with the addition of a saturated solution of hydrogenchloride in methanol. The reaction was triturated with 3 ml. ofmethanol, filtered an the filtrate was diluted with 40 ml. of acetone,yield 75 mg.

EXAMPLE 27 Preparation of 2-methylcyclopentyl BM123γ

A solution of 211 mg. of BM123γ, 3 ml. of 2-methylcyclopentanone and 98mg. of sodium cyanoborohydride in 50 ml. of methanol as stored at roomtemperature for 3.5 hours. During this time the pH of the solution wasmaintained at 7 with the addition of a saturated solution of hydrogenchloride in methanol. The reaction was triturated with 3 ml. ofmethanol, filtered and the filtrate was diluted with 40 ml. of acetone,yield 157 mg.

We claim:
 1. A compound selected from the group consisting of those ofthe formulae: ##STR6## wherein R is a moiety selected from the groupconsisting of those of the formulae: ##STR7## R₁ is selected from thegroup consisting of hydrogen, lower alkyl, halo substituted lower alkyl,lower alkenyl, phenyl, acetamidophenyl, nitrophenyl, mercaptophenyl,anisyl, tolyl, phenyl lower alkyl, 2-furyl, methyl substituted 2-furyl,2thienyl, methyl substituted 2-thienyl, 2-pyrryl, methyl substituted2-pyrryl, 2-pyridyl and 2-quinolyl; and the pharmacologically acceptableacid-addition salts thereof.
 2. A mixture consisting essentially of acompound according to claim 1, formula (I) thereof, wherein R is moiety(V) and a compound according to claim 1, formula (I) thereof, wherein Ris moiety (VI).
 3. A mxiture consisting essentially of a compoundaccording to claim 1, formula (II) thereof, wherein R is moiety (V) anda compound according to claim 1, formula (II) thereof, wherein R ismoiety (VI).
 4. A mixture consisting essentially of a compound accordingto claim 1, formula (III) thereof, wherein R is moiety (V) and acompound according to claim 1, formula (III) thereof, wherein R ismoiety (VI).
 5. A mixture consisting essentially of a compound accordingto claim 1, formula (IV) thereof, wherein R is moiety (V) and a compoundaccording to claim 1, formula (IV) thereof, wherein R is moiety (VI). 6.The mixture in accordance with claim 2 wherein R₁ is hydrogen.
 7. Themixture in accordance with claim 2 wherein R₁ is methyl.
 8. The mixturein accordance with claim 2 wherein R₁ is benzyl.
 9. The mixture inaccordance with claim 2 wherein R₁ is 1,1-dimethylpropyl.
 10. Themixture in accordance with claim 2 wherein R₁ istrans-1-methylpropen-1-yl.
 11. The mixture in accordance with claim 3wherein R₁ is hydrogen.
 12. The mixture in accordance with claim 3wherein R₁ is methyl.
 13. The mixture in accordance with claim 4 whereinR₁ is hydrogen.
 14. The mixture in accordance with claim 4 wherein R₁ ismethyl.
 15. The mixture in accordance with claim 5 wherein R₁ ishydrogen.
 16. The mixture in accordance with claim 5 wherein R₁ ismethyl.
 17. A compound selected from the group consisting of those ofthe formula: ##STR8## wherein R is a moiety selected from the groupconsisting of those of the formulae: ##STR9## R₂ is selected from thegroup conisting of lower alkyl, halo substituted lower alkyl and phenyllower alkyl; R₃ is selected from the group consisting of lower alkyl,halo substituted lower alkyl, lower alkenyl, lower cycloalkyl, phenyl,acetamidophenyl, nitrophenyl, mercaptophenyl, anisyl, tolyl, phenyllower alkyl, acetamidophenyl lower alkyl, nitrophenyl lower alkyl,mercaptophenyl lower alkyl, anisyl lower alkyl and tolyl lower alkyl;and R₂ and R₃ taken together with the associated methylidyne group iscyclopentyl, mono-lower alkyl substituted cyclopentyl, di-lower alkylsubstituted cyclopentyl, tri-lower alkyl substituted cyclopentyl,cyclohexyl, mono-lower alkyl substituted cyclohexyl, di-lower alkylsubstituted cyclohexyl or tri-lower alkyl substituted cyclohexyl; andthe pharmacologically acceptable acid-addition salts thereof.
 18. Amixture consisting essentially of a compound according to claim 17wherein R is moiety (A) and a compound according to claim 17 wherein Ris moiety (B).
 19. The compound according to claim 17 wherein R ismoiety (A), R₂ is methyl, and R₃ is methyl.
 20. The compound accordingto claim 17 wherein R is moiety (B), R₂ is methyl, and R₃ is methyl. 21.The mixture in accordance with claim 18 wherein R₂ is methyl and R₃ ismethyl.
 22. The compound according to claim 17 wherein R is moiety (A),R₂ is ethyl, and R₃ is methyl.
 23. The compound according to claim 17wherein R is moiety (B), R₂ is ethyl, and R₃ is methyl.
 24. The mixturein accordance with claim 18 wherein R₂ is ethyl and R₃ is methyl. 25.The compound according to claim 17 wherein R is moiety (A), R₂ is ethyl,and R₃ is ethyl.
 26. The compound according to claim 17 wherein R ismoiety (B) R₂ is ethyl, and R₃ is ethyl.
 27. The mixture in accordancewith claim 18, wherein R₂ is ethyl and R₃ is ethyl.
 28. The mixture inaccordance with claim 18 wherein R₂ is methyl and R₃ is n-propyl. 29.The mixture in accordance with claim 18 wherein R₂ is methyl and R₃ isbenzyl.
 30. The mixture in accordance with claim 18 wherein R₂ is methyland R₃ is 2-methylpropyl.
 31. The mixture in accordance with claim 18wherein R₂ is methyl and R₃ is isopropyl.
 32. The mixture in accordancewith claim 18 wherein R₂ is methyl and R₃ is 2,2-dimethylpropyl.
 33. Themixture in accordance with claim 18 wherein R₂ is ethyl and R₃ is butyl.34. The mixture in accordance with claim 18 wherein R₂ is methyl and R₃is 1-methylpropyl.
 35. The mixture in accordance with claim 18 whereinR₂ is methyl and R₃ is 2-phenylethyl.
 36. The mixture in accordance withclaim 18 wherein R₂ is methyl and R₃ is 3-methylbutyl.